Experimental characterization of next-generation expanded-bed adsorbents for capture of a recombinant protein expressed in high-cell-density yeast fermentation.

Expanded-bed adsorption (EBA) can be particularly useful in protein recovery from high-cell-density fermentation broth where conventional methods for harvest and clarification, such as continuous centrifugation and depth filtration, demand long processing times and are associated with high costs. In this work, the use of next-generation high-particle-density EBA adsorbents, including two mixed-mode resins, for the direct capture of a recombinant protein expressed in yeast at high cell densities is evaluated. Using classical experimental approaches and under different conditions (pH, salt, etc.), Langmuir isotherm parameters for these resins are obtained along with pore diffusivity values. Additional batch adsorption studies with Fastline® MabDirect, the resin that demonstrated the highest static binding capacity for the recombinant protein of interest under the conditions evaluated in this study, indicate competitive binding of nontarget proteins and approximately a 30% reduction in equilibrium binding capacity to 50 mg/mL settled bed in the presence of a 5%-10% cell concentration. Packed-bed (PB) dynamic binding capacities for the MabDirect resin (25-40 mg/mL PB) were significantly higher than for the Fastline® HSA resin and for the MabDirect MM resin in expanded-bed mode (5-10 mg/mL settled bed). Bed expansion behavior for the mMabDirect MM resin along with process yield and eluate purity are identified as a function of linear velocity and cell density, demonstrating process feasibility for pilot scale use.

Simultaneous Monitoring and Comparison of Multiple Product Quality Attributes for Cell Culture Processes at Different Scales Using a LC/MS/MS Based Multi-Attribute Method.

In response to the FDA's call for applying Quality by Design (QbD) to the manufacturing process, the biopharmaceutical industry has invested extensively into the monitoring and controlling of product quality attributes for bioprocesses. To assure the safety and efficacy of the drug product, defining critical quality attributes (CQA) and understanding their correlation with critical process parameters (CPP) becomes vitally important. In this work, a liquid chromatography-mass spectrometry based multi-attribute method (MAM) has been applied to the monitoring and trending of multiple CQAs of a monoclonal antibody product. To the best of our knowledge, this is the first demonstration of applying MAM to both a 3-liter development mini-bioreactor (3 L bioreactor) and a 2000-liter GMP single use bioreactor (2000L SUB). MAM was proven not only to be a great analytical tool for monitoring product quality attributes throughout the time course of the cell culture process, it could also provide critical product quality information in order to understand any potential process performance differences during scale-up and/or technology transfer. The successful monitoring and trending of the multiple CQAs throughout the 17-day cell culture process lays a solid foundation for possible real time in-process control and release of biotherapeutics using MAM in the future.

Two-component protein adsorption kinetics in porous ion exchange media.

This work provides a theoretical analysis of multicomponent adsorption kinetics for conditions typical of protein adsorption in porous ion exchangers as well as experimental results for the adsorption of lysozyme/cytochrome c mixtures in the cation exchanger SP-Sepharose-FF. The theory predicts the formation of overshoots in the intraparticle concentration profiles and in the total amount adsorbed for the more weakly adsorbed component. An analytical solution valid for the case where the isotherms are rectangular is developed and found to be in good agreement with the limiting behavior of the general numerical solution of the model equations. The experimental results show that the two proteins are competitively adsorbed and that an overshoot of adsorbed cytochrome c occurs during simultaneous adsorption. Model predictions based on the assumption that the adsorption isotherms are rectangular and that lysozyme completely displaces cytochrome c are in qualitative and quantitative agreement with the experimental kinetics suggesting that the overshoot phenomena observed with multicomponent systems in these resins can be explained with a diffusion model without the need to account for flux coupling or electrophoretic contributions to transport.

Radiotracer measurements of protein mass transfer: kinetics in ion exchange media.

We describe a method to measure protein mass transfer kinetics in ion exchange adsorbents for preparative chromatography based on the use of radioactively labeled protein. The method was developed and evaluated using lysozyme as a test protein with the three commercial strong-acid cation exchangers SP-Sepharose-FF, SP-Sepharose-XL, and S-HyperD. Iodination with 125I was used to label the protein, which was added in trace amounts (approximately 0.1%) to an unlabeled protein solution. The solution was recirculated through a shallow bed of the adsorbent particles and the radioactivity accumulated in the bed measured with a gamma-counter as a function of time. Radiotracer-based kinetics measurements were found to be in good agreement with results obtained with a conventional shallow-bed technique, provided that freshly labeled protein solutions were used. The method has advantages in terms of simplicity, ability to deal with adsorption from complex mixtures, and the potential for measurements under tracer diffusion conditions. Kinetics results obtained for the three different stationary phases were generally consistent with previous studies. Protein mass transfer can be described by a pore diffusion model with a nearly salt-independent pore diffusivity for SP-Sepharose-FF and by a homogeneous diffusion model with a saltindependent adsorbed phase diffusivity for S-HyperD. However, it appears that a more complex model, accounting for parallel pore and surface diffusion, is needed to describe protein mass transfer in SP-Sepharose-XL. The modeling results were found to be correlated with the apparent pore sizes determined by inverse SEC.